Mouse anti-Human, Mouse ZAP70 Monoclonal Antibody | anti-ZAP70 antibody

Anti-ZAP70 Purified

• Reactivity: Human, Mouse
• Applications: Flow Cytometry, Functional Assay, Immunoprecipitation, Western Blot, Immunocytochemistry
• Purity: Purified by protein-A affinity chromatography
• Synonyms: ZAP70; Monoclonal Antibody; Anti-ZAP70 Purified; ZAP-70; ADMIO2; zeta-chain (TCR) associated protein kinase; anti-ZAP70 antibody

• Host: Mouse
• Reactivity: Human, Mouse
• Clonality: Monoclonal
• Isotype: Mouse IgG1 kappa
• Clone Number: 1E7.2
• Specificity: The mouse monoclonal antibody 1E7.2 recognizes ZAP70, a 70kDa protein tyrosine kinase expressed in T and NK cells (an intracellular antigen). ZAP70 is a molecule susceptible to degradation. It is recommended to use freshly prepared cell lysates (protease inhibitors are essential) to avoid non-specific staining of degradation products. The epitope is located within "interdomain B".
• Purity/Purification: Purified by protein-A affinity chromatography
• Form/Format: Purified. Phosphate buffered saline (PBS) solution with 15mM sodium azide
• Concentration: 1mg/ml (varies by lot)

• Applicable Applications for anti-ZAP70 antibody: Flow Cytometry (FC/FACS) (QC tested), Immunoprecipitation (IP), Western Blot (WB), Immunocytochemistry (ICC)
• Application Notes: FC/FACS: Recommended dilution for purified antibody: 1ug/ml, intracellular staining.
• Immunogen: A KLH-coupled peptide corresponding to amino acids 282-307 of human ZAP70
• Notes: Recommended protocol for primary antibody conjugates using ADG Fix and Perm kit: 1) Add 50 ul peripheral blood to a 5 ml FACS tube; 2) Proceed surface staining with appropriate amount of surface antibody (20 min at room temperature); 3) Add 100 ul of Reagent A (Fixation Medium, stored and used at room temperature); 4) Incubate for 15 minutes at room temperature; 5) Add 4 ml PBS buffer and centrifuge at 300 g for 5 minutes at room temperature; 6) Remove supernatant and add to cells pellet 100 ul Reagent B (Permeabilization Medium) and 20 ul of ZAP-70-PE antibody or 10 ul of ZAP-70-FITC antibody (red blood cells are not lysed yet); 7) Vortex at low speed for 1-2 seconds (pellet must be resuspended); 8) Incubate for 15 minutes at room temperature; 9) Add to cells mixture of 1 ml Reagent B and 3 ml PBS buffer and centrifuge at 300 g for 5 minutes at room temperature; 10) Remove supernatant and resuspend cells in 100 ul PBS buffer; 11) Analyze immediately.
• Preparation and Storage: Store at 2-8 degree C. Do not freeze. Do not use after expiration date stamped on the label.

Western Blot (WB)

(Western blotting analysis of human ZAP70 using mouse monoclonal antibody 1E7.2 on lysates of MOLT-4 cell line and HEK293T/17 cell line (ZAP70 non-expressing cell line; negative control) under non-reducing and reducing conditions. Nitrocellulose membrane was probed with 2 ug/ml of mouse anti-ZAP70 monoclonal antibody 1E7.2 followed by IRDye800-conjugated anti-mouse IgG1 secondary antibody. A specific band was detected for ZAP70 protein at approximately 70 kDa.)

Related Product Information for anti-ZAP70 antibody

The ZAP70 (zeta-associated protein of 70kDa) tyrosine kinase was identified as a tyrosine phosphoprotein that associates with TCR zeta subunit and undergoes tyrosine phosphorylation following TCR stimulation. ZAP70 is a Syk family tyrosine kinase primarily expressed in T and NK cells that plays an essential role in signaling through the TCR. TCR-mediated activation of T cells is crucial to the immune response. In humans, ZAP70 gene mutations resulting in lower ZAP70 protein expression levels or expression of catalytically inactive ZAP70 proteins, have been identified. ZAP70 deficiency results in the absence of mature CD8+ T cells and the prevention of TCR-mediated activation of CD4+ T cells, and it can lead to severe combined immunodeficiency.In patients with chronic lymphocytic leukemia (B-CLL), ZAP70 expression on B cell was shown to be correlated with disease progression and survival. ZAP70 contains two N-terminal SH2 domains (Src homology domain 2) and a C-terminal kinase domain. During T cell activation, the binding of ZAP-70 SH2 domains to the phosphorylated zeta subunit on the activated TCR complex causes a colocalization with the Lck tyrosine kinase that phosphorylates ZAP70 on Tyr493 in the activation loop. ZAP70 autophosphorylates multiple tyrosines in the region between the SH2 domains and the kinase domain, including the binding sites for additional SH2-containing signaling proteins such as SLP76, LAT, Lck, PLCgamma1, Vav, Shc, Ras-GAP, and Abl. ZAP70-mediated activation of these downstream effectors leads to the release of intracellular calcium stores, and the transcription of interleukin-2 and other genes important for an immune response.

References

*Rossi FM, Del Principe MI, Rossi D, Irno Consalvo M, Luciano F, Zucchetto A, Bulian P, Bomben R, Dal Bo M, Fangazio M, Benedetti D, Degan M, Gaidano G, Del Poeta G, Gattei V: Prognostic impact of ZAP-70 expression in chronic lymphocytic leukemia: mean fluorescence intensity T/B ratio versus percentage of positive cells. J Transl Med. 2010 Mar 8;8:23., URL: http://www.ncbi.nlm.nih.gov/pubmed/20211015,*Vroblova V, Vrbacky F, Hrudkova M, Jankovicova K, Schmitzova D, Maly J, Krejsek J, Smolej L: Significant change in ZAP-70 expression during the course of chronic lymphocytic leukemia. Eur J Haematol. 2010 Jun;84(6):513-7., URL: https://www.ncbi.nlm.nih.gov/pubmed/20132303,*Sargent RL, Craig FE, Swerdlow SH: Comparison of Bcl-2, CD38 and ZAP-70 Expression in Chronic Lymphocytic Leukemia. Int J Clin Exp Pathol. 2009 Jun 16;2(6):574-82., URL: https://www.ncbi.nlm.nih.gov/pubmed/19636404,*Gachard N, Salviat A, Boutet C, Arnoulet C, Durrieu F, Lenormand B, Leprêtre S, Olschwang S, Jardin F, Lafage-Pochitaloff M, Penther D, Sainty D, Reminieras L, Feuillard J, Béné MC: Multicenter study of ZAP-70 expression in patients with B-cell chronic lymphocytic leukemia using an optimized flow cytometry method. Haematologica. 2008 Feb;93(2):215-23., URL: https://www.ncbi.nlm.nih.gov/pubmed/18223290,*Preobrazhensky SN, Bahler DW: Optimization of flow cytometric measurement of ZAP-70 in chronic lymphocytic leukemia. Cytometry B Clin Cytom. 2008 Mar;74(2):118-27., URL: https://www.ncbi.nlm.nih.gov/pubmed/17948236,*Del Principe MI, Del Poeta G, Buccisano F, Maurillo L, Venditti A, Zucchetto A, Marini R, Niscola P, Consalvo MA, Mazzone C, Ottaviani L, Panetta P, Bruno A, Bomben R, Suppo G, Degan M, Gattei V, de Fabritiis P, Cantonetti M, Lo Coco F, Del Principe D, Amadori S: Clinical significance of ZAP-70 protein expression in B-cell chronic lymphocytic leukemia. Blood. 2006 Aug 1;108(3):853-61., URL: https://www.ncbi.nlm.nih.gov/pubmed/16601244,*Letestu R, Rawstron A, Ghia P, Villamor N, Boeckx N, Boettcher S, Buhl AM, Duerig J, Ibbotson R, Kroeber A, Langerak A, Le Garff-Tavernier M, Mockridge I, Morilla A, Padmore R, Rassenti L, Ritgen M, Shehata M, Smolewski P, Staib P, Ticchioni M, Walker C, Ajchenbaum-Cymbalista F: Evaluation of ZAP-70 expression by flow cytometry in chronic lymphocytic leukemia: A multicentric international harmonization process. Cytometry B Clin Cytom. 2006 Jul 15;70(4):309-14., URL: https://www.ncbi.nlm.nih.gov/pubmed/16906588

NCBI and Uniprot Product Information

• NCBI GeneID: 22637
• UniProt Accession #: P43404
• UniProt Protein Name: Tyrosine-protein kinase ZAP-70
• Protein Family: Tyrosine-protein kinase
• UniProt Gene Name: Zap70
• UniProt Synonym Gene Names: Srk; Zap-70
• UniProt Entry Name: ZAP70_MOUSE

Uniprot Description

ZAP70: a tyrosine kinase of the Syk family. Associates with the T-cell antigen receptor zeta-chain after TCR stimulation. Phosphorylated by Src-family kinases following antigen receptor activation. Plays a role in lymphocyte activation.

Protein type: Kinase, protein; Protein kinase, tyrosine (non-receptor); EC 2.7.10.2; Protein kinase, TK; TK group; Syk family

Cellular Component: T cell receptor complex; extrinsic to internal side of plasma membrane; membrane; cytoplasm; plasma membrane; immunological synapse; intracellular; intercellular junction; cytosol; lipid raft

Molecular Function: transferase activity; protein binding; phosphotyrosine binding; protein-tyrosine kinase activity; transferase activity, transferring phosphorus-containing groups; nucleotide binding; non-membrane spanning protein tyrosine kinase activity; kinase activity; ATP binding; receptor binding; protein kinase activity

Biological Process: thymic T cell selection; cell migration; peptidyl-tyrosine phosphorylation; immune system process; protein amino acid autophosphorylation; positive regulation of calcium-mediated signaling; protein amino acid phosphorylation; T cell receptor signaling pathway; negative thymic T cell selection; positive regulation of T cell differentiation; innate immune response; positive regulation of alpha-beta T cell proliferation; immune response; beta selection; phosphorylation; transmembrane receptor protein tyrosine kinase signaling pathway; positive regulation of alpha-beta T cell differentiation; positive thymic T cell selection